Oxidative phosphorylation, electron transport and active transport of amino acids are all membrane related phenomena which share common properties. Nevertheless, the basic properties of each process provides insight into their bioenergetic mechanisms. Some essential differences in these processes were observed with membrane vesicles differing in spatial orientation. Although protoplast ghost (right side out) membranes from Mycobacterium phlei were capable of oxidation, their ability to couple phosphorylation was cryptic. The addition of a soluble protein fraction to intact ghosts resulted in the transfer of the energy rich phosphate bond to exogenous nucleotides. In the presence of the soluble protein and exogenous ADP, ghost preparations preloaded with C14-ADP failed to yield exogenous C14-ATP, although ATP was formed. In contrast, ghost preparations preloaded with P32-ADP resulted in the formation of exogenous P32-ATP on addition of the soluble protein. It would appear that the soluble fraction is involved in the translocation of the energy rich phosphate bond. Active transport of prolines in the ghosts and electron transport particles have been compared and characterized. Membrane vesicles (oriented inside out) depleted of membrane-bound coupling factors (CF) lacked latent ATPase but were capable of active transport of amino acids and substrate oxidation. Removal of the membrane-bound CF resulted in a collapse of the proton gradient but failed to affect active transport of amino acids. Treatment of the depleted membrane vesicles with phospholipase A resulted in membrane conformational changes and in a 50% loss of active transport of proline; however, the addition of cardiolipine restored active transport. In contrast, phospholipase A treatment of the intact membrane vesicles failed to inhibit active transport. These results indicate that inside out oriented membranes may be involved in active transport.